The major observation of Project 3 in the current Program Project is that mice heterozygous for glutathione peroxidase 4 (Gpx4) have a significant increase in life span compared to wild type (WT) mice. This increase in life span occurs despite Gpx4+/- mice showing reduced expression of Gpx4 in all tissues throughout their life span;embryonic fibroblasts and liver from Gpx4+/- mice showing increased sensitivity to oxidative stress; and the levels of oxidative damage not being significantly altered in tissues of Gpx4+/- mice. We believe that the Gpx4+/- mouse model is a valuable new model for studying the mechanism of aging because the increase in life span of these mice appears to occur through a novel pathway. For example, there is no evidence that the increased life span of these mice arises either from increased resistance to oxidative stress/reduced oxidative damage or from alterations in insulin/IGF-l signaling, two pathways associated with increased longevity in invertebrates and rodents. We hypothesize that reduced Gpx4 expression increases longevity through a mechanism that involves alterations in the mitochondrialpathway ofapoptosis, leading to an increased sensitivity of cells to the induction ofapoptosis and an enhanced removal of damaged cells from the organism. If we confirm our hypothesis in this project, it will be the first demonstration that enhanced apoptosis can have an anti-aging action. We propose to test our hypothesis by the following Specific Aims. 1. To measure the sensitivity of mice deficient in either mitochondrial or cytosolic Gpx4 to the induction of apoptosis by oxidative stress. Transgenic mice expressing either the mitochondrial form of Gpx4 (mtGpx4) or the cytosolic form of Gpx4 (cytGpx4) will be crossed to Gpx4+/- mice to generate mice deficient in either mtGpx4 or cytGpx4. Cells and tissues of mice deficient in either mtGpx4 or cytGpx4 will be exposed to various types of oxidative stress and the induction of apoptosis, CL levels and oxidation, and the release of pro-apoptotic factors from mitochondria will be followed with age and compared to WT mice. 2. To measure the ability of mice deficient in either mitochondrial or cytosolic Gpx4 to remove damaged cells, specifically, cells that can give rise to tumors. The mutant frequency and the number of tumors in tissues from mice deficient in either mtGpx4 or cytGpx4 will be measured with age and compared to WT mice. 3. To measure the survival and age-sensitive biomarkers of mice deficient in either mitochondrial or cytosolic Gpx4.